Elevated levels of atmospheric carbon dioxide (CO 2 ) can directly affect the cold hardening process in evergreens through their effect on the accumulation of carbon and nitrogen reserves. This study investigated the biochemical responses of black spruce [ Picea mariana (Mill.) B.S.P.] seedlings to CO 2 enrichment during growth, cold hardening and dehardening. Seedlings were grown under 350 (ambient) or 710 (elevated) ppm of CO 2 for 12 months in eight mini-greenhouses. Photoperiod and temperature were gradually lowered in autumn to induce cold hardening, and the conditions were reversed in spring to promote dehardening. At regular intervals, cold tolerance was assessed and sugars, starch and amino acid concentrations were measured. The freezing tolerance differed between the two treatments only in early autumn, with seedlings growing under high CO 2 being more tolerant. The northern ecotype was more cold tolerant with concomitant higher concentrations of sucrose, fructose, pinitol, glucose and total soluble sugars. The concentration of soluble sugars increased in needles and roots of black spruce along with cold hardening, and the concentrations of the cryoprotective sugars sucrose and raffinose were lower under elevated CO 2 . Amino acid concentrations were also lower under elevated than under ambient CO 2. The lower level of reserve did not translate into a lower level of freezing tolerance.